Essential role of the mitochondrial apoptosis-inducing factor in
programmed cell death
Nicholas Joza*², Santos A. Susin³, Eric Daugas³§, William L. Stanfordk, Sarah K. Cho², Carol Y. J. Lik, Takehiko Sasaki*¶, Andrew J. Elia*, H.-Y. Mary Cheng*¶, Luigi Ravagnan³, Karine F. Ferri³, Naoufal Zamzami³, Andrew Wakeham*, Razqallah Hakem*, Hiroki Yoshida*, Young-Yun Kong*, Tak W. Mak*, Juan Carlos ZuÂnÄiga-P¯uÈcker², Guido Kroemer³ & Josef M. Penninger*²¶
* Amgen Institute, 620 University Avenue, Toronto, Ontario, Canada M5G 2C1
³ Centre National de la Recherche Scienti®que, UMR1599, Institut Gustave Roussy, 39 rue Calmette Desmoulins, F-94805 Villejuif, France
§ Assistance Publique, HoÃpitaux de Paris, Service de NeÂphrologie B, HoÃpital Tenon, 20 rue de la Chine, F-75020 Paris, France k Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5
¶ Ontario Cancer Institute, and the Departments of Medical Biophysics, and ² Immunology, University of Toronto, Toronto, Ontario, Canada M5S 1A1
...
Programmed cell death is a fundamental requirement for embryogenesis, organ metamorphosis and tissue homeostasis. In mammals, release of mitochondrial cytochrome c leads to the cytosolic assembly of the apoptosomeÐa caspase activation complex involving Apaf1 and caspase-9 that induces hallmarks of apoptosis. There are, however, mitochondrially regulated cell death pathways that are independent of Apaf1/caspase-9. We have previously cloned a molecule associated with programmed cell death called apoptosis-inducing factor (AIF). Like cytochrome c, AIF is localized to mitochondria and released in response to death stimuli. Here we show that genetic inactivation of AIF renders embryonic stem cells resistant to cell death after serum deprivation.
Moreover, AIF is essential for programmed cell death during cavitation of embryoid bodiesÐthe very ®rst wave of cell death indispensable for mouse morphogenesis. AIF-dependent cell death displays structural features of apoptosis, and can be genetically uncoupled from Apaf1 and caspase-9 expression. Our data provide genetic evidence for a caspase-independent pathway of programmed cell death that controls early morphogenesis.
Programmed cell death (PCD) is a fundamental property of all multicellular organisms. It is crucial for plant and animal develop- ment, insect and amphibian metamorphosis, organ morphogenesis, tissue homeostasis, ageing, and the removal of infected or damaged cells1. The biochemical and ultrastructural features of apoptosis are highly conserved throughout the evolution of multicellular animals1±4. PCD has been linked to the CED9/Bcl-2, CED4/Apaf1 and CED3/caspase-9 genes that are essential for PCD in Caenorhabditis elegans and vertebrates5±8. In response to death stimuli, mitochondrial membranes are permeabilized9,10, and cytochrome c is released from mitochondria3,11,12 and associates with Apaf1 and pro-caspase-9 to trigger a caspase activation cascade that culminates in cell death characterized by apoptotic morphology7,13±15. Failure to invoke appropriate cell death can result in cancer or autoimmunity, whereas increased PCD can lead to degenerative processes such as immunode®ciency and neurodegen- erative disease16.
Although the cytochrome c/Apaf1/caspase-9 apoptosome is essential for several PCD pathways, cells de®cient in these molecules can still die3. Indeed, cytochrome c, apaf1 and caspase-9 knockout mouse embryos undergo normal, albeit delayed, morphogen- esis17±21. Moreover, cell lines derived from these mutant mice are not uniformly resistant to death stimuli, but instead undergo PCD in a manner speci®c to both cell type and death signal19. It has also been shown that Bcl-2 preserves the integrity of mitochondrial membranes and protects cells from death independently of Apaf1 and caspases, implying that Bcl-2 interferes with two different mitochondrion-dependent death effector cascades22,23. Thus, a death effector system other than cytochrome c/Apaf1/caspase-9 must be able to induce PCD.
We previously cloned apoptosis-inducing factor (AIF), which, like cytochrome c, is normally present in the mitochondrial inter- membrane space and is released in response to death stimuli24,25.
Extramitochondrial targeting of AIF, micro-injection of recombi- nant AIF protein into cells, or addition of AIF to isolated nuclei leads to the generation of apoptotic phenotypes, such as chroma- tin condensation and phosphatidylserine exposure on the cell surface24. AIF has also been implicated in the control of apoptosis in syncytia induced by the HIV type-1 envelope glycoprotein26, indicating that AIF may be involved in the pathogenesis of HIV infections. But although AIF can induce certain aspects of cell death in cultured cells, whether it is essential for PCD in vivo remains unresolved. Moreover, AIF has never been linked to PCD at the genetic level.
To explore the role of AIF in the control of PCD during animal development, we disrupted the mouse aif gene by homologous recombination. We report here that AIF is essential for the ®rst wave of PCD required for embryonic morphogenesis and cavitation.
Moreover, inactivation of AIF renders embryonic stem cells resis- tant to cell death after serum starvation. These results provide the
®rst genetic evidence of a second, mitochondrially regulated cell death pathway in mammalian cells that is critical for morphogenesis and PCD after withdrawal of survival factors.
Gene targeting of aif in embryonic stem cells
The murine aif gene was ablated in embryonic stem (ES) cells using a targeting vector that deleted exon 3, corresponding to the amino terminus of the mature protein (nucleotides 247±346, amino acids 83±115). Three independent aif-targeted ES cell clones were obtained. Because the aif gene maps to the X chromosome24, mutation of one aif allele resulted in a complete knockout in XY male ES cells and absence of aif expression by northern and western blotting (see Supplementary Information Fig. 1). As a control for changes to ES cells during G418 selection, ES cell clones were isolated in which the neomycin resistance cassette had integrated randomly into the genome (aifneo/Y).
Three independent aif-/YES cell clones were injected into C57BL/
6 blastocysts to generate chimaeric mice, and into rag1-/-blastocysts for lymphocyte reconstitution27. Whereas all parental wild-type ES cell clones (aif+/Y) and all aifneo/YES cell clones could contribute to adult tissues in chimaeric mice and reconstitute T- and B-cell lineages in rag1-/- mice, we failed to observe any chimaerism using all three aif-/YES cells clones. Using in vitro ES cell differ- entiation and formation of teratocarcinoma-like tumours in vivo28, however, aif-/YES cell clones differentiated into cells from all three germ layers, including cartilage, muscle, neuronal tissue, epithe- lium, B cells, myeloid and erythroid cells (see Supplementary Information Fig. 1)29. Thus, aif-/YES cells retain their capacity to differentiate into cells from all three germ layers.
aif-/YES cells are resistant to growth factor deprivation The aif-/Y ES cell lines exhibited normal proliferation in vitro.
Unlike cytochrome c-/-, apaf1-/- and caspase-9-/- ES cells17,19,21, aif2 =Y ES cell lines displayed normal susceptibility to death, which was preceded by the dissipation of the mitochondrial transmembrane potential (Dwm), in response to staurosporine, etoposide, azide, tert-butylhydroperoxide (Fig. 1a), anisomycin or ultraviolet irradiation (data not shown). This normal susceptibility to cell-death induction was observed both in the absence and in the presence of the pan-caspase inhibitor Z-VAD.fmk (Fig. 1a).
Whereas serum withdrawal results in cell death of aif+/Y, aifneo/Y and apaf1-/-ES cells23, all three aif2 =YES cell lines largely conserved their viability and normal mitochondrial membrane integrity (Dwm) when cultured in the absence of serum (Fig. 1b). Moreover, in the presence (but not the absence) of Z-VAD.fmk, aif-/YES cell lines failed to die in response to the pro-apoptotic agent vitamin K3
(menadione) (Fig. 1b). Thus, AIF is rate-limiting for some pathways of death induction. In particular, aif-/YES cells are resistant to death after growth factor withdrawal.
AIF is essential for cavitation of embryoid bodies
The absence of overt chimaerism in whole organisms, yet the apparently normal differentiation potential of aif-/Y ES cells in vitro and in vivo, suggested that AIF might be required for normal
t-BHP Azide
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aif+/yaif–/y
aif–/y
aif–/y +z-VAD aif+/y +z-VAD DiOC6(3)low
Figure 1 AIF is essential for cell death induced by serum withdrawal. a, Susceptibility of aif-/YES cells to the death stimuli staurosporine (STS), etoposide (Etop.), azide and tert- butylhydroperoxide (t-BHP). ES cells were cultured in 10% serum in the presence of the indicated lethal stimuli and stained with propidium iodide (PI; for cell viability) and DiOC6
(3) (for mitochondrial Dwm). Note that all PI+cells are DiOC6(3)low. Mean values 6 s.e.m.
for three control and three aif-/Ycell lines are shown. b, Resistance of aif-/YES cells to cell death induced by menadione and serum withdrawal. FACS data are shown for aif+/Yand a representative aif-/YES cell clone. Numbers are mean values 6 s.e.m. for three control and three different aif-/Ycell lines in the corresponding quadrants.
% EBs
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aif+/Y simple
aif+/Y cyst aif–/Y (3) simple aif–/Y (2) simple
aif–/Y (3) cyst aif–/Y (2) cyst
Figure 2 AIF is essential for embryoid body cavitation. a, Morphology (rows 1, 3) and histology (rows 2, 4; haematoxylin and eosin (HE) stain) of simple EBs (day 3), cystic (cavitated) EBs (day 6), and expanded cysts (day 14) derived from a wild-type (aif+/Y) and an aif-/YES clone. Note the complete block in cavitation in the absence of AIF expression.
Scale bars, 100 mm. b, Percentages at day 21 of ES cell aggregates, simple and cystic EBs from one aif+/Y, two aifneo/Yand three aif-/YES cell clones. At least 250 EBs were counted per genotype. c, Kinetics of EB formation. Shown is the frequency of simple and cystic EBs on days 6, 12 and 21.
PCD during early embryonic development. PCD occurs throughout mammalian development, beginning with apoptosis of the initially solid embryonic ectoderm to generate the proamniotic cavity30. This early developmental process can be mimicked in vitro by culturing aggregates of ES cells in the absence of leukaemia inhibitory factor and feeder cells31. Under these culture conditions, ES cells form undifferentiated cell aggregates that develop into simple embryoid bodies (EBs), de®ned as multicellular aggregates containing an outer layer of endodermal cells and a solid core of undifferentiated ectodermal cells (Fig. 2a, left). The inner cells of simple EBs subsequently undergo PCD to form cystic EBs (Fig.
2a, top centre), a process called cavitation. As cystic embryoid bodies are cultured in vitro, the cavity expands (Fig. 2a, top right). The removal of cells of the inner core to form a cavitated or cystic EB is the ®rst known wave of PCD during mouse morphogenesis30.
When aif-/YES cells were tested in the EB formation assay, they were able to form simple EBs at frequencies and with kinetics comparable to those of aif+/Yand aifneo/Y controls (Fig. 2). But whereas a signi®cant proportion of aif+/Yand aifneo/YEBs underwent cavitation to form cystic EBs, EBs from all three differentiated aif2 =YES cell lines exhibited a complete block in cavitation (Fig. 2a, b; and Supplementary Information Fig. 2). As cavitation is essential for the initiation of gastrulation and thus subsequent steps in embryogenesis32, defective cavitation by aif-/Y EBs probably explains the inability of aif-/Y ES cells to lead readily to adult tissue in chimaeric mice.
AIF controls PCD during early morphogenesis
Impaired cavitation might be due to either increased proliferation and/or impaired PCD of the cells that form the inner core. To
investigate whether EB cell proliferation was increased in the absence of AIF, we examined BrdU (5-bromodeoxyuridine) incor- poration by wild-type and aif-/YEBs. Although aif-/YEBs displayed abnormal morphology (Fig. 3a, left) and histology (Fig. 3b, left), no evidence was obtained for increased proliferation of the inner cells of aif-/YEBs at day 3, day 5, or at any later time point as compared with wild-type EBs (data not shown). To assay for PCD, the inner cells from wild-type and aif-/YEBs were analysed by DAPI (49,6- diamidino-2-phenylindole dihydrochloride) staining to detect chromatin condensation (Fig. 3c, left) and by assays for in situ caspase-3 activation (Fig. 3d, left). Massive apoptosis was observed in the wild-type EBs, but no signs of cell death were found among the inner cells of aif-/YEBs. These results indicate that impaired cavitation in aif-/YEBs is not caused by enhanced proliferation but is due to a failure of inner cells to undergo PCD.
The outer endoderm cells have been suggested to provide death signals to inner cells required for cavitation30; however, histological and electron microscopy analyses showed that simple aif-/YEBs do not lack endodermal tissue. Furthermore, aif-/YEBs expressed the endoderm-speci®c32,33markers BMP2, BMP4, GATA-4, a-fetopro- tein and HNF-4 (data not shown). To establish that the defects of aif-/Ycells are autonomous to inner cells, we generated chimaeric EBs by mixing aif-/Yand wild-type ES cells expressing a lacZ reporter (aif+/Y; lacZ) (Fig. 4a). Although cavitation was partially rescued in these chimaeric EBs (Fig. 4b), cell death was restricted to wild-type
a
b
c
d
aif+/Y aif–/Y apaf1–/– casp-9–/–
Figure 3 AIF is essential for PCD during early morphogenesis. a, b, Morphology (a) and histology (b; HE stain) of day 14 EBs from aif+/Yand aif-/YES cells (left), and apaf1-/-and caspase-9-/-ES cells (right). Note lack of cavitation in aif-/YEBs but normal cavitation in apaf1-/-and caspase-9-/-EBs. Scale bars, 100 mm. c, d, Apoptotic features of inner cells from day 6 EBs from aif+/Yand aif-/YES cells (left), and apaf1-/-and caspase-9-/-ES cells (right). c, DAPI staining (blue) to visualize chromatin condensation (arrows). d, Caspase-3 activation. Note absence of chromatin condensation and caspase-3 activation in aif-/YEBs. Similar results were obtained for two other aif-/YES clones and at later time points (up to day 21). All results for aifneo/YEBs (n = 3) paralleled those obtained for wild-type EBs. Scale bar, 20 mm (c); 100 mm (d).
% cell death
a
c
e
b
d
f
aif+/Y;lacZ aif–/Y
0 20 40 60 80
Figure 4 The effect of AIF is autonomous to inner cells. aif-/YES cells and wild-type cells expressing lacZ (aif+/Y; lacZ) were mixed to generate chimaeric EBs. a±e, At day 4 a or day 9 (b±e), EBs were stained with X-gal to mark lacZ-expressing cells (blue) and counterstained with nuclear fast red. c, d, High-magni®cation views of b showing viable aif-/Yinner cells (pink only) (c) and dead wild-type inner cells (blue) (d). e, Quanti®cation of death in inner cells from day 9 chimaeric EBs. From each of 6 distinct EBs, 80±100 inner cells were counted. Mean values 6 s.e.m. are shown. f, aif-/Ycells can differentiate into columnar epithelium (asterisk). Scale bar, 100 mm (a, b); 10 mm (c). Arrows in b±d and f indicate dead cells (fragmented nuclei and/or presence of cellular debris).
(blue) inner cells (Fig. 4b±e). Our mixing experiments also showed that aif-/Ycells can differentiate into columnar epithelium (Fig. 4f).
These results indicate that impaired cavitation in aif-/YEBs is not caused by defective endoderm formation. Instead, impaired cavita- tion is due to an intrinsic failure of AIF-de®cient inner cells to undergo PCD.
Apaf1 and caspase-9 are not required for cavitation
The death of inner cells in wild-type EBs was found to be accom- panied by the activation of caspase-3 (Fig. 3d, left), an effector caspase downstream of the cytochrome c/Apaf1/caspase-9 apopto- some. We therefore explored the contribution of Apaf1 and caspase- 9 to cavitation by analysing the development of EBs from apaf1-/- and caspase-9-/-ES cells17,19. Genetic inactivation of the apaf1 and caspase-9 genes abolished caspase-3 activation (Fig. 3d, right).
However, loss of Apaf1 or caspase-9 expression had no apparent effect on cavitation (Fig. 3a, b; and Supplementary Information Fig. 3) or the death of inner cells (Fig. 3c). The kinetics and extent of cells that undergo chromatin condensation were comparable among wild-type, apaf1-/-and caspase-9-/-EBs (n = 5 per group). Adding the broad-spectrum caspase inhibitor z-VAD.fmk to developing wild-type EBs also failed to block cell death and subsequent cavitation. These results show that the PCD required for EB cavitation can occur in the absence of caspase-3 activation and can be genetically uncoupled from Apaf1 and caspase-9.
We next examined the intracellular localization of AIF in EBs and the effect of mutations of apaf1, caspase-9 or aif on AIF and cytochrome c mobilization. In response to death stimuli AIF translocates from the mitochondria to the nucleus, whereas cyto- chrome c localizes to the cytosol24. AIF (Fig. 5a, red colour) was found to translocate from the mitochondria to the nucleus (green DNA stain) in inner cells, but not in the outer endodermal cells of wild-type, apaf1-/- and caspase-9-/- EBs. Cytochrome c was also released from mitochondria of wild-type, apaf1-/-and caspase-9-/- inner cells (Fig. 5b). There was no detectable cytochrome c translo- cation from mitochondria to the cytosol in inner cells of aif-/YEBs, indicating that mitochondrial membranes fail to permeabilize. This result is consistent with the failure of aif-/Yinner cells to activate caspase-3 (Fig. 3d), a defect that presumably results from de®cient assembly of the apoptosome. These ®ndings indicate that AIF acts upstream of cytochrome c and independently of the cytochrome c/Apaf1-triggered caspase activation cascade during cavitation.
AIF-regulated PCD has characteristic features of apoptosis It has been reported that cell death of apaf1-/-and caspase-9-/-ES cells in response to ultraviolet radiation exhibits the morphological features of necrosis rather than apoptosis19,23. To establish whether AIF-controlled cell death in EBs has the ultrastructural character- istics of apoptosis, inner cells from wild-type and aif-/YEBs were compared using electron microscopy. Dying inner cells in wild-type a
b
aif+/Y
aif+/Y aif–/Y apaf1–/– casp-9–/–
aif–/Y
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20 µm 100 µm
Figure 5 Translocation of AIF from the mitochondria to the nucleus. a, Immunolocalization of AIF (red) in outer and inner cells of day 6 wild-type, apaf1-/-, caspase-9-/-and aif-/Y EBs. In viable cells, AIF is sequestered in mitochondria (punctate red spots) separated from the nucleus (DNA-binding dye Sytox Green). In dying inner cells, AIF translocates from mitochondria to nuclei and green/red overlap appears yellow. Insets show high magni®cations. b, Immunolocalization of cytochrome c (red) in inner cells of day 6 EBs.
Cytochrome c (punctate staining in viable cells) accumulates in the cytosol in wild-type, caspase-9-/-and apaf1-/-cells undergoing PCD (diffuse staining). Cytochrome c is retained in mitochondria of aif-/Ycells.
a
c
e
v
v
b
d
f
Figure 6 Morphological features of AIF-regulated PCD in EBs. Electron micrographs of inner ectodermal cells of day 6 wild-type (a), aif-/Y(b), apaf1-/-(c) and
caspase-9-/-(d±f) EBs. A viable inner cell appears in aif-/YEB (b). Note chromatin condensation (asterisks in a and c±f), plasma membrane blebbing (arrows in c, d), and preserved structural integrity of mitochondria (arrowheads in a, c, f) and of cytosolic organelles such as endoplasmic reticula (`.' in c, f). e, Formation of apoptotic bodies.
Original magni®cation, ´6,000 (a±d); ´15,000 (e); ´20,000 (f).
EBs displayed typical apoptotic morphology (Fig. 6a), including the presence of chromatin condensation, plasma membrane blebbing, formation of apoptotic bodies, and a preserved ultrastructure of cytoplasmic organelles. Inner cells from aif-/YEBs retained a healthy phenotype (Fig. 6b). Intriguingly, the inner cells from both caspase- 9-/-and apaf1-/-EBs exhibited typical features of apoptosis, such as intact nuclear and plasma membranes, chromatin condensation (Fig. 6c±f, asterisks), plasma membrane blebbing (Fig. 6c, d, arrows), formation of apoptotic bodies (Fig. 6e), and preserved ultrastructure of mitochondria (Fig. 6c, f, solid arrowheads) and rough endoplasmic reticula (Fig. 6c, f, `.'). These features were similar to those in wild-type inner cells.
Consistent with the absence of caspase-3 activation, caspase-9-/- and apaf1-/-EBs do not manifest an advanced pattern of chromatin compaction (Fig. 6a). Instead, a peripheral type of chromatin compaction predominated (Fig. 6c±f). Thus, with the exception of caspase-dependent advanced chromatin compaction, the AIF- regulated pathway of PCD required for embryonic cavitation exhibits classical ultrastructural features of apoptosis and is inde- pendent of the Apaf1/caspase-9-mediated PCD pathway.
Discussion
In C. elegans, genetic evidence suggested that apoptosis is strictly dependent on caspase activation5. We provide genetic evidence here that not all apoptosis of mammalian cells is dependent on caspases, and that an AIF-dependent, caspase-independent PCD pathway exists that is crucial for cell death following growth factor depriva- tion and early mammalian development.
AIF and mitochondrial control of apoptosis. Numerous reports have shown that caspase inhibition prevents mammalian cell death or blocks the acquisition of morphological and biochemical char- acteristics of apoptosis34. Moreover, mutational analyses of cyto- chrome c (ref. 21), caspases19,20and Apaf1 (ref. 17) showed that these molecules contribute to apoptosis in a manner speci®c to both cell type and death signal. Similarly, aif-/YES cells, unlike apaf1-/-and caspase-9-/-ES cells, are sensitive to various apoptotic stimuli, such as staurosporine, anisomycin, ultraviolet irradiation and etoposide.
However, aif-/YES cells are resistant to serum withdrawal and AIF is essential for the ®rst wave of cell death during mouse morphogen- esis. These data indicate the coexistence of two separate pathways linking the mitochondria to apoptosis, one that requires AIF and the other that relies on caspase activation.
The results of our study provide de®nitive genetic evidence that AIF inactivation abolishes all signs of cell death in early morpho- genesis, including the mitochondrial release of cytochrome c.
Moreover, AIF is a rate-limiting factor of ES cell death induced by menadione (only if caspases are simultaneously blocked) or by serum withdrawal (independently of caspase inhibition), indicating a stimulus-dependent contribution of AIF to the apoptotic cascade.
The exact hierarchies and communication between AIF and the cytochrome c/Apaf1/caspase-9 apoptosome in cell-type- and death- signal-speci®c PCD remain to be determined.
Morphogenesis of multicellular organisms. PCD is essential during early animal development for the sculpting of digits, the palate and the eyes, the formation of hollow organs and the neural tube, and the generation of sexual organs1. In early mouse embryos, the proamniotic cavity is formed by the death of the ectodermal cells in the core of the developing embryo30. Thus, PCD is an integral part of morphogenesis and metamorphosis at all stages of animal development. Because developmental PCD exhibits the structural hallmarks of apoptosis, the ®nding that C. elegans bearing muta- tions of their caspase (CED-3) or Apaf1 (CED-4) orthologues have normal lifespans2was originally surprising. Moreover, morphogen- esis and organ sculpting are also normal in cytochrome c, apaf1 and caspase-9 knockout mouse embryos, albeit delayed17±21. These observations pointed to the existence of another PCD pathway that can compensate for the absence of caspase-dependent apoptosis and
that is highly conserved through evolution. As AIF messenger RNA and protein expression can be detected throughout murine embryo- genesis and in all developing organs (see Supplementary Informa- tion Fig. 4), it is likely that AIF contributes to morphogenesis at later stages of embryogenesis.
We have shown that the genetic inactivation of AIF abolishes the
®rst wave of developmental cell death occurring during early mouse embryogenesis. Assuming that ontogenesis recapitulates phylogeny, it is tempting to speculate that AIF represents a pathway of apoptosis that predates the caspase pathway. Whereas AIF homo- logues have been found in all metazoan phyla35, no evidence for caspases has been reported in plants, fungi or unicellular organisms such as the Trypanosoma cruzi epimastigote, all of which can nevertheless undergo PCD1. We propose that AIF and the AIF- regulated cell death pathway constitute an ancient and conserved process required for the morphogenesis of multicellular organisms.
The identi®cation of the molecules involved in this PCD pathway and their genetic and functional characterization should yield new insights into the basic physiology of cell death, and might allow us to develop strategies for the modulation of the cell death
machinery. M
Methods
aif-de®cient ES cells and chimaeric mice
The aif gene was cloned from a 129/SVJ mouse genomic library using a mouse aif probe (nucleotides 247±346). A targeting vector (600 base pairs short arm, and 6 kilobases long arm) ¯anking a PGK-Neo cassette was electroporated into male E14K ES cells. ES cell colonies resistant to G418 (300 mg ml-1) were screened for homologous recombination by polymerase chain reaction (sense primer, 59-GGGATTAGATAAATGCCTGCTCTT-39;
antisense primer, 59-CCCCCAAACTTATATCAGCCTACCTTC-39). Recombinant colonies were con®rmed by Southern blotting of HindIII-digested genomic DNA hybridized to a ¯anking probe. Total RNAwas extracted from aif-/YES cells and subjected to northern blotting using full-length AIF complementary DNA. Absence of AIF protein in aif-/YES cells was determined by western blotting using an antibody reactive to residues 151±200 of murine AIF24. Antibodies to Apaf1 (Upstate Biotechnology) and actin (Sigma) were used as controls. To test contribution to adult tissues, aif-/YES cells were injected into blastocysts from rag1-/-mice27and C57BL/6 mice to generate chimaeric animals. Mice were maintained at the animal facilities of the Ontario Cancer Institute in accordance with institutional guidelines. Equivalent results and phenotypes were obtained for three independent aif-/YES cell clones. Apaf1-/-and caspase-9-/-ES cells have been described17,19.
ES cell differentiation
Parental wild-type aif+/Y, three aif-/YES cell clones and ES cell clones in which Neo was randomly integrated (aifneo/Y) were cultured under conditions promoting differentiation into EBs29,36. Chimaeric EBs were generated using aif-/YES cell clones (lacZ-negative) and aif+/YES cells constitutively expressing lacZ (aif+/Y; lacZ). The aif+/Y; lacZ ES cell clone contains a randomly integrated copy of the lacZ gene fused to the chicken b-actin promoter37. For colony assays, single EB cell suspensions were replated in methylcellulose.
Blood islands were detected using benzidine. ES cells were further differentiated into primitive mesodermal cells by co-culture with OP9 bone marrow stromal cells for 5 d.
Single-cell suspensions from these cultures were either used for FACS analysis of Flk1+ hemangioblasts or replated onto OP9 cells and grown for an additional 5±12 d. Colonies were counted 10±14 d later and stained with Wright±Giemsa to analyse morphology, and with anti-CD45, CD11b, CD19 and TER-119 monoclonal antibodies38. In vivo tumour formation of ES cells in athymic nu/nu mice and detection of differentiated tissues have been described28.
Quanti®cation of cell death
We cultured ES cells in the presence of 10% fetal calf serum and leukaemia inhibitory factor. Cell death was induced by addition of staurosporine (2 mM, 24 h), etoposide (100 mM, 24 h), sodium azide (15 mM, 48 h), tert-butylhydroperoxide (200 mM, 48 h) or menadione (150 mM, 24 h), or by serum withdrawal (0%, 72 h), in the presence or absence of Z-VAD.fmk (50 mM). Death was quanti®ed by staining with propidium iodide (PI;
5 mg ml-1) and DiOC6(3) (40 nM).
Immunostaining and in situ procedures
For in situ localization of AIF and cytochrome c (ref. 25), paraformaldehyde-®xed EBs were stained with rabbit antiserum raised against residues 151±200 of AIF, or anti- cytochrome c monoclonal antibody (clone 6H2.B4, Pharmingen) followed by PE- conjugated goat anti-rabbit IgG (anti-AIF) or PE-conjugated goat anti-mouse IgG (anti- cytochrome c). Cells were counterstained with 10 nM Sytox Green (Molecular Probes).
Staining was detected by confocal scanning ¯uorescence microscopy. Electron microscopy and in situ DAPI staining to detect chromatin condensation were as described19. Activated
caspase-3 was detected using an antibody speci®c for the cleaved (active) form of caspase-3 (New England BioLabs). In situ hybridization of murine embryos at distinct stages of development was done using sense and antisense probes from murine aif cDNA (nucleotides 456±1607).
Received 1 November 2000; accepted 31 January 2001.
1. Jacobson, M. D., Weil, M. & Raff, M. C. Programmed cell death in animal development. Cell 88, 347±
354 (1997).
2. Vaux, D. L. & Korsmeyer, S. J. Cell death in development. Cell 96, 245±254 (1999).
3. Green, D. R. & Reed, J. C. Mitochondria and apoptosis. Science 281, 1309±1312 (1998).
4. Kerr, J. F., Wyllie, A. H. & Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239±257 (1972).
5. Ellis, H. M. & Horvitz, H. R. Genetic control of programmed cell death in the nematode C. elegans.
Cell 44, 817±829 (1986).
6. Hengartner, M. O. & Horvitz, H. R. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell 76, 665±676 (1994).
7. Zou, H., Henzel, W. J., Liu, X., Lutschg, A. & Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405±413 (1997).
8. Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M. & Horvitz, H. R. The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell 75, 641±652 (1993).
9. Newmeyer, D. D., Farschon, D. M. & Reed, J. C. Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 353±364 (1994).
10. Kroemer, G. & Reed, J. C. Mitochondrial control of cell death. Nature Med. 6, 513±519 (2000).
11. Kluck, R. M., Bossy-Wetzel, E., Green, D. R. & Newmeyer, D. D. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275, 1132±1136 (1997).
12. Yang, J. et al. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science 275, 1129±1132 (1997).
13. Liu, X., Kim, C. N., Yang, J., Jemmerson, R. & Wang, X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147±157 (1996).
14. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479±489 (1997).
15. Zou, H., Li, Y., Liu, X. & Wang, X. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11549±11556 (1999).
16. Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456±1462 (1995).
17. Yoshida, H. et al. Apaf1 is required for mitochondrial pathways of apoptosis and brain development.
Cell 94, 739±750 (1998).
18. Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. A. & Gruss, P. Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727±737 (1998).
19. Hakem, R. et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339±352 (1998).
20. Kuida, K. et al. Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325±337 (1998).
21. Li, K. et al. Cytochrome c de®ciency causes embryonic lethality and attenuates stress-induced apoptosis. Cell 101, 389±399 (2000).
22. Amarante-Mendes, G. P. et al. Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell death. Cell Death Differ. 5, 298±306 (1998).
23. Haraguchi, M. et al. Apoptotic protease activating factor 1 (Apaf-1)-independent cell death suppression by Bcl-2. J. Exp. Med. 191, 1709±1720 (2000).
24. Susin, S. A. et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441±446 (1999).
25. Daugas, E. et al. Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. FASEB J. 14, 729±739 (2000).
26. Ferri, K. F. et al. Apoptosis control in syncytia induced by the HIV type 1-envelope glycoprotein complex. Role of mitochondria and caspases. J. Exp. Med. 192, 1081±1092 (2000).
27. Chen, J., Lansford, R., Stewart, V., Young, F. & Alt, F. W. RAG-2-de®cient blastocyst complementation:
an assay of gene function in lymphocyte development. Proc. Natl Acad. Sci. USA 90, 4528±4532 (1993).
28. Martin, G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634±7638 (1981).
29. Bautch, V. L. et al. Blood island formation in attached cultures of murine embryonic stem cells.
Dev. Dyn. 205, 1±12 (1996).
30. Coucouvanis, E. & Martin, G. R. Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83, 279±287 (1995).
31. Robertson, E. J. Embryo-Derived Stem Cell Lines (IRL, Oxford, 1987).
32. Coucouvanis, E. & Martin, G. R. BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development 126, 535±546 (1999).
33. Leahy, A., Xiong, J. W., Kuhnert, F. & Stuhlmann, H. Use of developmental marker genes to de®ne temporal and spatial patterns of differentiation during embryoid body formation. J. Exp. Zool. 284, 67±81 (1999).
34. McCarthy, N. J., Whyte, M. K., Gilbert, C. S. & Evan, G. I. Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell Biol. 136, 215±227 (1997).
35. Lorenzo, H. K., Susin, S. A., Penninger, J. & Kroemer, G. Apoptosis inducing factor (AIF): a phylogenetically old, caspase-independent effector of cell death. Cell Death Differ. 6, 516±524 (1999).
36. Schmitt, R. M., Bruyns, E. & Snodgrass, H. R. Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev. 5, 728±740 (1991).
37. Shalaby, F. et al. Failure of blood-island formation and vasculogenesis in Flk-1-de®cient mice. Nature 376, 62±66 (1995).
38. Cho, S. K. et al. Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl Acad. Sci. USA 96, 9797±9802 (1999).
Supplementary information is available on Nature's World-Wide Web site (http://www.nature.com) or as paper copy from the London editorial of®ce of Nature.
Acknowledgements
We thank M. Saunders for scienti®c editing; A. Oliveira-dos-Santos, K. Bachmaier, T. Wada, V. Stambolic, L. Zhang, M. Crackower, C. Krawzcyk, I. Kozieradzki, Q. Liu, J. Irie-Sasaki, M. Nghiem, R. Sarao, E. Grif®th, L. Barra and A. Manoukian for comments;
D. MeÂtivier and B. Calvieri for technical assistance; and J. Rossant and A. Bernstein for lacZ-expressing ES cells. N.J. and J.M.P. are supported by the Canadian Institute of Health Research (CIHR), Amgen, and the National Cancer Institute of Canada. W.L.S. is supported by the Karyn Glick Memorial Special Fellowship and CIHR. E.D. is supported by Assistance Publique-HoÃpitaux de Paris and CANAM. G.K. is supported by grants from Ligue Nationale Contre le Cancer, European Commission and Agence Nationale pour la Recherche Sur le SIDA. J.M.P. holds a Canadian Research Chair in Cell Biology.
Correspondence and requests for materials should be addressed to J.M.P.
(e-mail: jpenning@amgen.com).
